Phospholipases C (EC 3.1.4.3) are a family of enzymes which hydrolyze the sn-3 phosphodiester bond in membrane phospholipids producing diacylglycerol and a phosphorylated polar head group. Mammalian phospholipase C (PLC) enzymes exhibit specificity for the polar head group which is hydrolyzed, i.e., phosphatidylcholine, phosphatidylinositol, etc. Recently, much interest has been generated in those PLC enzymes which selectively hydrolyze phosphoinositide lipids in response to receptor occupancy by agonist. Hydrolysis of phosphatidylinositol 4,5-bisphosphate generates two second messenger molecules; diacylglycerol, a co-factor required for activation of protein kinase C, and inositol 1,4,5-trisphosphate, a soluble second messenger molecule which promotes the release of intracellular nonmitochrondrial stores of calcium (Berridge, Ann. Rev. Biochem., 56:159-193, 1987). The diacylglycerol released may be further metabolized to free arachidonic acid by sequential actions of diglycerol lipase and monoglycerol lipase. Thus, phospholipases C are not only important enzymes in the generation of second messenger molecules, but may serve an important role in making arachidonic acid available for eicosanoid biosynthesis in select tissues.
Mammalian tissues contain multiple distinct forms of phosphoinositide-specific PLC (Crooke and Bennett, Cell Calcium, 10:309-323, 1989; Rhee et al., Science, 244:546-550, 1989). It is proposed that each of the enzymes couple to distinct classes of cell surface receptors, i.e., PLC-.alpha. couples to vasopressin receptors, PLC-.gamma. couples to growth factor receptors, etc. (Aiyar et al., Biochem. J., 261:63-70, 1989; Crooke and Bennett, Cell Calcium, 10:309-323, 1989; Margolis et al., Cell, 57:1101-1107, 1989; Wahl et al., Proc. Natl. Acad. Sci. USA, 86:1568-1572, 1989). PLC.gamma..sub.1 contains src-homology regions (SH2 and SH3) that appear to mediate the interaction between the enzyme and receptors with tyrosine kinase activity, such as the epidermal growth factor (EGF) receptor (Stahl et al. Nature, 332:269-272 (1988); Katan et al., Cell, 54:171-177 (1988)).
It has been established that a rapid synthesis of prostaglandins (PG) from arachidonic acid in macrophages usually accompanies inflammatory stimuli. Thus, inhibition of the release of arachidonic acid from macrophages would provide an effective control of PG synthesis and thereby inflammatory conditions. Recently, phospholipase C has been characterized as an enzyme which is involved in the biosynthetic phosphatidylinositol-arachidonic acid-prostaglandin pathway. This finding is further substantiated by the observation that phospholipase C is inhibited by phenothiazine, a compound known to inhibit the stimulated release of arachidonic acid from macrophages and prostaglandins from platelets.
PLC.gamma. is the only isozyme that is phosphorylated by activated tyrosine kinase growth factor receptors (Rotin et al., EMBO J., 11:559-567 (1992); Mohammadi et al., Mol. Cell. Biol., 11:5068-5078 (1992); Kim et al., Cell, 65:435-441 (1991)). Following growth factor stimulation, cytosolic PLC.gamma. is extensively and rapidly phosphorylated in vivo (50-70% of the PLC.gamma. molecules are modified within 5 minutes). This phosphorylation apparently induces the relocation of PLC.gamma. to the plasma membrane where presumably it is better able to interact with its phospholipid substrates. In vitro studies utilizing enzyme that had previously been immunoprecipitated from cells suggest that the catalytic activity of the phosphorylated form of PLC.gamma..sub.1 is increased compared to that of the unphosphorylated form, although this effect also depends on the assay conditions. These results suggest that PLC.gamma. may be an important component of mitogenic signal transduction. Furthermore, altered PLC.gamma. activity may correlate with some disease states. For example, an increase in the concentration of PLC.gamma. has been documented in cells derived from primary human breast carcinomas which also overexpress the EGF receptor (Arteaga et al., Proc. Natl. Acad. Sci. U.S.A., 88, 10435-10439 (1991 )). Thus, inhibition of PLC.gamma. activity, particularly of the activated form, may be of therapeutic value in the treatment of breast cancer.
All mammalian tissues which have been studied exhibit one or more PI-PLC enzymes. Generally, more than one enzyme exists in a single mammalian cell type. PI-PLC enzymes do exhibit tissue selectivity in their distribution. PLC-.gamma. is found predominantly in neural tissues and is the major enzyme in the brain. However, isolation of the enzyme from mammalian tissue involves cumbersome organic solvent extractions and only limited quantities of enzyme can be obtained in this way. No information is available concerning the genetic regulation of PI-PLC enzymes, mRNA or protein stability.
To date, the cDNA for 6 distinct PI-PLC enzymes have been cloned. The enzymes range in size from 504 amino acids to 1250 amino acids, and are remarkably divergent considering that they exhibit similar biochemical properties. Four of the five enzymes (PLC-.beta., PLC-.delta.1, PLC-.delta.2, and PLC-.alpha.) contain two domains approximately 250 amino acids in length which exhibit between 50 to 80% sequence similarity. PLC-.alpha. contains sequences with 35% similarity to the first domain only (Crooke and Bennett, Cell Calcium, 10:309-323, 1989). The marked differences in DNA sequences for the different PI-PLC enzyme allows the selective targeting of one PI-PLC enzyme, without affecting other enzymes using antisense technology. The human cDNA clone has been reported for PLC-.delta..sub.1 and PLC-.delta..sub.2, (Ohta et al., FEBS Lett., 242:31-35, 1988) and PLC-.gamma.1 (Burgess et al., Mol. Cell. Biol., 10:4770-4777 (1990)). The rest are rat cDNA clones. The genomic clones have not been reported for any of the PI-PLC enzymes.
Expression and purification of phospholipase C-.delta..sub.1 as a fusion protein in E. coli has been previously reported (R. Ginger and P. Parker, Eur. J. Biochem., 210:155-160(1992). The expression and purification of rat phospholipase C-.delta..sub.1 in E. coli has also been reported by M. Ellis et al. (Eur. J. Biochem., 213:339-347(1993)), however the expression system utilized resulted in either largely insoluble protein or such a low expression that the specific activity of the full length enzyme could not be determined. There are reports of expression of rat phospholipase C-.gamma. in E. coli where no purification of the enzyme was attempted (Y. Emori et al., J.Biol. Chem . . , 264:21885-21890 (1989)).
The cloning of bovine brain phospholipase C.gamma. has been previously reported (M. L. Stahl et al., Nature, 332:269-272 (1988)) and the enzyme has been expressed in mammalian cells. Proof of the expression in mammalian cells was only by PLC activity of the cell lysates and the enzyme was never isolated.
Accordingly, it is an object of this invention to provide a convenient process for the preparation of phospholipase C.gamma. which is isolated in an active soluble form.
It is also the object of this invention to provide a cDNA construct which can be used to express such a phospholipase C.gamma..
Another object of this invention is to provide a convenient assay for phospholipase C.gamma. activity which can be used to identify inhibitors of such activity.